Verocytotoxin (VT) is an Escherichia coli produced toxin which has been shown to be involved in the aetiology of hemolytic uremic syndrome (HUS), the leading cause of pediatric renal failure. Some strains of E. coli elaborate cytotoxins that are active on cultured vero cells. At least two of the verocytotoxins, verocytotoxin 1 and verocytotoxin 2, are known to be produced by E. coli strains that are closely associated with a non-specific diarrheal illness, as well as two distinct complications, the hemolytic uremic syndrome (HUS) and hemorrhagic colitis. A third verocytotoxin SLTII, distinct from VT2, has also been identified and other related cytotoxins are becoming recognized, but have yet to be fully characterized.
VT1 is closely related, both antigenically and biologically to Shiga Toxin produced by Shigella dysenteriae type 1 and is thus also referred to as Shiga-like toxin (SLT). It has been reported that the cytotoxin purified from S. dysenteriae type 1 binds to glycosphingolipids containing the Gal(.alpha.1-4)Gal sequence in a terminal position [Lindberg, A.A. et al (1986) in Protein Carbohydrate Interactions in Biological Systems, Lark D., ed. pp 439-446 Academic Press, London] although residual binding to globotetraosyl ceramide was observed. Keusch has recently confirmed that Shiga toxin binds specifically to Gb0se.sub.3 cer but maintains that this is a nonproductive binding in relation to cytotoxicity, since the addition of chiotriose protects against cytotoxicity with little effect on Gb0se.sub.3 cer binding [Jacewicz, M. et al, (1986) J. Exp. Med. 163 pp 1391-1404] although this result was not confirmed [Brown, J. E. et al, Annual Meeting of the American Society of Microbiologists, Las Vegas, Nev. Abstr. B107, p. 36]. Furthermore, there has yet been no discussion of the role of the lipid portions of these glycolipids in the receptor binding of either Shiga-toxin or verocytotoxin.
In E. coli, verocytotoxins are encoded by one or more bacteriophages and, furthermore, individual strains may produce either one or both VT1 and VT2. Both the natural and the recombinant forms of E. coli verocytotoxin have been isolated. One such recombinant cloned toxin is pJLB28 which expresses both the A and B subunits [Huang, A. et al. (1986) J. Bacteriol. 166, 375-379].
E. coli verocytotoxin has been characterized as having an "A" subunit of approximately 31,000 daltons and several "B" subunits each having an approximate molecular weight of 5,500 daltons. The A subunit possesses the biological activity of the toxin which is involved in inhibiting protein synthesis, whereas the B subunits are presumed to mediate specific binding and receptor-mediated uptake of the toxin.
At present, verocytotoxin is detected by a tedious and time consuming (but highly sensitive) procedure involving the determination of cytotoxicity to cells in culture. This procedure requires extensive cell culture facilities, the availability of toxin-neutralizing antibodies and thus considerable technical expertise. The assay is therefore performed in only a very few centers throughout the world. In fact, the assay is available only in reference and research laboratories. Moreover VTEC (verocytotoxin producing E. coli) may be a minor fraction of the intestinal flora and thus many colonies must be grown up and tested to exclude the possibility of these infections.
The demand for this assay is very high particularly in light of recent well publicized North American outbreaks in nursing homes, children's day care centers etc. Prospective studies in Alberta and Washington State have shown that VTEC are significant causes of endemic cases of hemorrhagic colitis and HUS. In Washington population based studies have shown that VTEC cause about 75% of sporadic cases of HUS. While E. coli 0157:H7 is the most common strain of VTEC, it is clear that HUS is caused by VTEC of many different serotypes. It has also become apparent that VTEC are a significant veterinary problem since they produce hemorrhagic colitis in calves. The oedema disease toxin which is produced by organisms found in pigs has also been shown to be a verocytotoxin and VTEC have been isolated from meat purchased at retail food outlets. Thus the need has also been strongly expressed for the screening of perishable food products for contamination with bacteria which produce this toxin. With improved assay technologies, such screening could be done at the place of manufacture.
Straightforward ELISA assays have so far not had the specificity or sensitivity required of such an assay. Part of the reason for this lies in the extreme biological potency of this toxin being effective at the picogram level.